Specific Aims Notch receptors anchor a fundamental signaling pathway conserved from sea urchins to humans. Signals transduced by these receptors normally influence cell fate decisions during development, and regulate cell growth, differentiation, and death in a variety of tissue types [7-9]. Notch signals are used iteratively at different decision points and have functional outcomes that depend heavily on gene dose and context. Both deficiencies and abnormal increases of Notch signaling are associated with human developmental anomalies and cancer, emphasizing the importance of precisely regulating Notch signal strength [1,10-15]. Notch receptors normally reside on the cell surface in a resting conformation, which is maintained by a negative regulatory region (NRR) that encompasses the LNR repeats and a heterodimerization (HD) domain immediately external to the membrane. Ligand binding induces proteolytic sensitivity at a metalloprotease cleavage site within the HD domain, with proteolysis at this site triggering subsequent cleavage by gamma-secretase to release the intracellular portion of Notch (ICN) from the membrane. After gamma-secretase cleavage, ICN translocates to the nucleus, and induces transcription of target genes by driving the assembly of a Notch transcriptional activation complex (NTC) that includes a DNAbound transcription factor called CSL (for CBF-1, Suppressor of Hairless, and Lag-1; [16]) and a coactivator protein of the Mastermind-like (MAML) family [17,18]. This CSL/ICN/MAML complex represents the central effector of Notch signaling, which then recruits additional factors through the C-terminal portion of MAML, like p300/CBP, PCAF/GCN5, and the CDK8/mediator complex [19-22] to drive target gene transcription. The overarching goal of these studies is to uncover the factors that govern the differing sensitivities of Notch target genes to transcriptional activation in different developmental, physiologic, and pathophysiologic contexts. To address this issue, we have combined biochemical, molecular, and structural approaches to gain insight into the mechanism of transcriptional activation. In the previous period of grant support, we mapped the portions of human MAML1, NOTCH1, and CSL required to assemble a core Notch transcription complex (NTC) on cognate DNA [23], and solved the structure of this complex by X-ray crystallography [24]. We then used insights from the structure to discover that Notch transcription complexes form dimers cooperatively on paired CSL binding sites found in a head-tohead arrangement in the promoters of certain Notch target genes [25]. Our findings identify a new mechanistic step in Notch signaling, and lead to the hypothesis that dimerization can act as a key regulatory event in the control of Notch target gene transcription. In the next period of support, we propose to examine this hypothesis, focusing on the structure and function of NTC dimers in transcriptional activation by pursuing the following specific aims: Aim 1. Elucidate the DNA binding-site preferences for NTC monomers and dimers. Aim 2. Determine the structure of an NTC dimer on a paired CSL binding site from the promoter of a Notch target gene. Aim 3. Investigate the functional implications of Notch dimerization in models of differentiation and disease pathogenesis.